Use of recombinant protein for treating metabolic disorders

ABSTRACT

Disclosed are methods and compositions useful in preventing or treating a metabolic disorder by using a protein having an amino acid sequence as set forth in SEQ ID NO: 1.

BACKGROUND OF THE INVENTION

Metabolic disorders such as type 2 diabetes mellitus (T2DM) and otherrelated complications are leading causes of mortality. These disordersare associated with the excessive nutritional intake and lack ofexercise of the Western lifestyle, which is increasing all over theworld. T2DM (and insulin resistant conditions) is a chronic,progressive, incompletely understood metabolic condition mainlycharacterized by hyperglycemia (a state of long-term elevated levels ofglucose in the blood). Impaired insulin secretion, resistance to tissueactions of insulin, or a combination of both are thought to be thecommonest reasons contributing to the pathophysiology of T2DM, aspectrum of disease originally arising from tissue insulin resistanceand gradually progressing to a state characterized by complete loss ofsecretory activity of the beta cells of the pancreas. Prolonged highblood sugar may cause blood vessel and nerve damage. The incidence oftype 2 diabetes is high and rising and is becoming a leading cause ofmortality, morbidity, and healthcare expenditure throughout the world.

Various pharmacological approaches for the treatment of T2DM areavailable. The major classes of oral antidiabetic medications includebiguanides, sulfonylureas, meglitinide, thiazolidinedione (TZD),dipeptidyl peptidase 4 (DPP-4) inhibitors, sodium-glucose cotransporter(SGLT2) inhibitors, and α-glucosidase inhibitors. However, the aboveapproaches have some side effects such as hypoglycemia and weight gain.

Accordingly, there is still a need for improved therapeutic approachesto metabolic disorders with fewer side effects.

SUMMARY OF THE INVENTION

It was unexpectedly found in the present invention that a recombinantprotein having an amino acid sequence as set forth in SEQ ID NO: 1(called “ES135” hereinafter) is effective in treating metabolicdisorders. For instance, when administering ES135 to a subject sufferingfrom a metabolic disorder, hyperglycemia, the level of blood glucose wassignificantly attenuated.

Accordingly, one aspect of the present invention is directed tocompositions for preventing or treating metabolic disorders, wherein thecompositions comprise ES135. Also provided herein are methods forpreventing or treating metabolic disorders comprising administrating aneffective amount of ES135.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the glucose-lowering effect of different dosages of ES135in C57BL/6 and db/db mice.

FIG. 2 shows the glucose-lowering effect of different dosages of ES135in C57BL/6 and ob/ob mice

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Provided herein are methods and compositions useful in preventing ortreating metabolic disorders by using ES135. Based on the studies indiabetic mouse model as described herein, the inventors have shown thatES135 is effective in attenuating the level of blood glucose as comparedto the vehicle group.

II. Definitions

The following abbreviations are used herein:

IM (i.m.): intramuscular injectionIV (i.v.): intravenous injectionIP (i.p.): intraperitoneal injectionSC (s.c.): subcutaneous injectionPO (p.o.): oral administrationICV (i.c.v.): intracerebroventricular injectionIT: intrathecal injectionBid: bis in die (twice daily)

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. Any methods, devices and materials similar orequivalent to those described herein can be used in the practice of thisinvention. The following definitions are provided to facilitateunderstanding of certain terms used frequently herein and are not meantto limit the scope of the present disclosure.

As used herein, the article “a” or “an” means one or more than one (thatis, at least one) of the grammatical object of the article, unlessotherwise made clear in the specific use of the article in only asingular sense.

As used herein, the term “ES135” refers to a recombinant proteincomprising an amino acid sequence of SEQ ID NO: 1, which was disclosedin U.S. Pat. No. 7,956,033, the content of which is hereby incorporatedby reference herein in its entirety. The amino acid sequence of SEQ IDNO:1 is as follows: Ala Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser AsnGly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly Thr Arg AspArg Ser Asp Gln His Ile Gln Leu Gin Leu Ser Ala Glu Ser Val Gly Glu ValTyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly LeuLeu Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu GluGlu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp PheVal Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr GlyGln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp. In someembodiments, the sequence of ES135 is at least 70%, 75%, 80%, 85%, 90%,95%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 1.In some embodiments, the amino acid sequence of SEQ ID NO: 1 has one ormore modifications. For example, the amino acid sequence of SEQ ID NO: 1has an N-terminal phosphogluconoylation or gluconoylation as disclosedin U.S. Pat. No. 9,567,385, the content of which is hereby incorporatedby reference herein in its entirety.

In one embodiment of the present invention, the amino acid sequence ofES135 consists of the amino acid sequence of SEQ ID NO: 1.

As used herein, the term “prevent”, “preventing” or “prevention” refersto precluding, averting, obviating, forestalling, stopping, or hinderingsomething from happening, especially by advance action.

As used herein, the term “treat”, “treating” or “treatment” refers toboth therapeutic treatment and prophylactic or preventative measures,wherein the object is to prevent or slow down (lessen) an undesiredphysiological condition, disorder or disease, or to obtain beneficial ordesired clinical results. For the purposes of this invention, beneficialor desired clinical results include, but are not limited to, alleviationof symptoms; diminishment of the extent of the condition, disorder ordisease; stabilization (i.e., not worsening) of the state of thecondition, disorder or disease; delay in onset or slowing of theprogression of the condition, disorder or disease; amelioration of thecondition, disorder or disease state; and remission (whether partial ortotal), whether detectable or undetectable, or enhancement orimprovement of the condition, disorder or disease. Treatment includeseliciting a clinically significant response without excessive levels ofside effects. Treatment also includes prolonging survival as compared toexpected survival if not receiving treatment.

As used herein, the term “therapeutically effective amount” or“effective amount” refers to an amount of ES135 to be used in thepresent invention which prevents, ameliorates or treats the symptomsaccompanying a disease or condition referred to in this specification,or provides a desirable therapeutic effect. Therapeutic efficacy andtoxicity of ES135 can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED50 (thedose therapeutically effective in 50% of the population) and LD50 (thedose lethal to 50% of the population). The dose ratio betweentherapeutic and toxic effects is the therapeutic index, and it can beexpressed as the ratio, LD50/ED50.

As used herein, the term “administering” when used in conjunction with atherapeutic means to administer a therapeutic directly into or onto atarget tissue or to administer a therapeutic to a patient whereby thetherapeutic positively impacts the tissue to which it is targeted. Thus,as used herein, the term “administering”, when used in conjunction witha compound, peptide or protein, can include, but is not limited to,providing a therapeutic into or onto the target tissue; providing atherapeutic systemically to a subject by, e.g., intravenous injectionwhereby the therapeutic reaches the target tissue. “Administering” acomposition may be accomplished by oral, injection, topicaladministration, or by either method in combination with other knowntechniques.

The term “animal”, “subject” or “patient” as used herein includes, butis not limited to, humans and non-human vertebrates such as wild,domestic and farm animals, preferably humans.

III. Embodiments of the Invention

In one aspect, the invention is directed to a pharmaceutical compositionfor preventing or treating a metabolic disorder in a subject, comprisinga protein having an amino acid sequence as set forth in SEQ ID NO: 1.

In another aspect, the invention provides a protein having an amino acidsequence as set forth in SEQ ID NO: 1, for use in the prevention ortreatment of a metabolic disorder in a subject.

In still another aspect, the invention provides use of a protein havingan amino acid sequence as set forth in SEQ ID NO: 1 in manufacturing amedicament for preventing or treating a metabolic disorder in a subject.

The metabolic disorder includes, but is not limited to, hyperglycemia,impaired fasting glucose, impaired glucose tolerance, insulinresistance, hyperinsulinemia, type I diabetes, type II diabetes,refractory diabetes, and combinations thereof. In some embodiments, themetabolic disorder is insulin resistance. Preferably, the metabolicdisorder is type II diabetes.

Hyperglycemia, or high blood sugar, can be defined as a fasting bloodglucose level higher than about 7, about 10, about 15, or about 20mmol/L. Specifically, hyperglycemia is defined as the condition in whicha subject has a fasting blood glucose concentration above the normalrange, greater than 110 mg/dL (6.11 mmol/L).

Impaired fasting glucose (IFG), is defined as the condition in which asubject has a fasting blood glucose concentration or fasting serumglucose concentration in a range from 100 to 125 mg/dL (i.e. from 5.6 to6.9 mmol/L), in particular greater than 110 mg/dL and less than 126mg/dL (7.00 mmol/L). A subject with “normal fasting glucose” has afasting glucose concentration smaller than 100 mg/dL, i.e. smaller than5.6 mmol/L.

Impaired glucose tolerance (IGT), which is a pre-diabetic state ofhyperglycemia, is defined as a two-hour glucose levels (glycemia) ofabout 140 to about 199 mg/dL (7.8 to 11.0 mmol) on the 75-g oral glucosetolerance test (according to WHO and ADA). Glycemia of about 200 mg/dLor greater is considered diabetes mellitus.

Insulin resistance is defined as a state in which a normal amount ofinsulin produces a subnormal biologic response. Insulin resistance canbe measured by the hyperinsulinemic euglycemic clamp technique,Homeostatic Model Assessment (HOMA), or Quantitative insulin sensitivitycheck index (QUICKI).

Hyperinsulinemia is defined as the condition in which a subject withinsulin resistance, with or without euglycemia, has fasting orpostprandial serum or plasma insulin concentration elevated above thatof normal, lean individuals without insulin resistance, having awaist-to-hip ratio <1.0 (for men) or <0.8 (for women). A fasting seruminsulin level greater than 25 mU/L or 174 pmol/L is consideredhyperinsulinemia. The term “euglycemia” is defined as the condition inwhich a subject has a fasting blood glucose concentration within thenormal range, greater than 70 mg/dL (3.89 mmol/L) and less than 110mg/dL (6.11 mmol/L).

Type I diabetes results from the body's failure to produce insulin, andhas also been called “insulin-dependent diabetes mellitus” (IDDM) or“juvenile diabetes”. Type II diabetes results from insulin resistance, acondition in which cells fail to use insulin properly, sometimescombined with an absolute insulin deficiency. It is also called “noninsulin-dependent diabetes mellitus” (NIDDM) or “adult-onset diabetes.”The defective responsiveness of body tissues to insulin is believed toinvolve the insulin receptor. Diabetes mellitus is characterized byrecurrent or persistent hyperglycemia, and in some examples diagnosed bydemonstrating any one of: a. Fasting plasma glucose level≥7.0 mmol/L(126 mg/dL); b. Plasma glucose≥11.1 mmol/L (200 mg/dL) two hours after a75 g oral glucose load as in a glucose tolerance test; c. Symptoms ofhyperglycemia and casual plasma glucose≥11.1 mmol/L (200 mg/dL); d.Glycated hemoglobin (Hb A1C)≥6.5%.

Refractory diabetes is characterized by poor glycaemic control despiteadequate treatment. In this condition, even the best therapeutic regimestailored for rapid symptomatic relief and attainment of glycaemic goalsmay not work. Many hypotheses, such as poor healthcare-seeking behaviorand lack of adherence on part of the patient, clinical inertia, andinappropriate choice of therapeutic regimes on part of the physician,and poor psychosocial support from family members or diabetes careproviders have all been suggested as reasons for this state. Asillustrated in the literature (J Diabetes. 2016 January; 8(1):76-85),early age of onset, longer duration of diabetes, greater complexity andnumber of therapies, use of insulin, and presence of microvascularcomplications are all predictors of refractory diabetes.

In one another aspect, the invention is directed to a method forpreventing or treating a metabolic disorder in a subject, comprisingadministering to the subject a therapeutically effective amount of aprotein having an amino acid sequence as set forth in SEQ ID NO: 1. Thetherapeutic effects of said protein have been proven in the diabeticanimal model as described herein.

In some embodiments, ES135 is administered in db/db and ob/ob diabeticanimal models for the evaluation of its glucose-lowering effect.Preferably, ES135 is administered subcutaneously (s.c.). In someembodiments, ES135 is administered once, twice, thrice per day or less,e.g., every second day, every third day, every week, every other week,or less. As demonstrated in the present invention, ES135 is administeredsubcutaneously once. The detailed study procedures are shown in theExamples below.

In some embodiments, administration of ES135 can normalize the level ofblood glucose in a subject. In db/db mice, when compared to the vehiclegroup, ES135 at 0.5 mg/kg SC significantly attenuated the blood glucoselevel (p<0.05) at 6^(th) and 30^(th) hr after treatment. ES135 at 1mg/kg SC also showed statistically significant attenuation of bloodglucose levels at 6^(th) hr, 18th hr, 30^(th) hr and 42^(th) hr aftertreatment. However, the combination group of ES135 at 0.2 mg/kg withheparin at 500 U/kg showed modest glucose-lowering effect in db/db miceduring the study period (FIG. 1)

In ob/ob mice, subcutaneous administration of ES135 at 0.5 mg/kg oncesignificantly attenuated the blood glucose level (p<0.05) at 6^(th) hrafter treatment compared to the vehicle group. ES135 at 1 mg/kg SC alsoshowed statistically significant attenuation of blood glucose levels at6^(th) hr, 18^(th) hr and 30^(th) hr after treatment, when compared tothe vehicle group. However, the combination group of ES135 at 0.2 mg/kgwith heparin at 500 U/kg showed modest glucose-lowering effect in ob/obmice during the study period (FIG. 2).

In C57BL/6 mice, subcutaneous administration of ES135 at 1 mg/kg oncedid not affect the blood glucose levels during the study period (FIGS. 1and 2).

In some embodiments, the dose of ES135 administered is equivalent to0.001-1 mg ES135 per kg body weight (i.e., 0.001-1 mg/kg) of thesubject, e.g., equivalent to 0.001-0.01, 0.01-0.05, 0.05-0.1, 0.1-0.2,0.1-0.4, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 or higher mg ES135 per kg bodyweight. As indicated in FIGS. 1 and 2, the glucose-lowering effect ofES135 in diabetic animal models is in a dose-dependent matter.Dose-dependent glucose-lowering effect in both db/db and ob/ob animalmodels was observed.

In conclusion, treatments of ES135 significantly attenuated theevaluated level of blood glucose.

According to the invention, ES135 may be constituted into any formsuitable for the mode of administration selected. Preferably, ES135 isadministered subcutaneously, topically, intraneurally, intravenously,intramuscularly, intracerebroventricularly, or intrathecally. Morepreferably, ES135 is administered subcutaneously.

EXAMPLES

The present invention is more specifically explained by the followingexamples. However, it should be noted that the present invention is notlimited to these examples in any manner.

Glucose-lowering effect after administration of ES135 in db/db and ob/obmice

ES135 was administered subcutaneously (0.2, 0.5 and 1 mg/kg) once togroups of 8 normal C57BL/6 mice, non-insulin dependent diabetic mellitus(NIDDM) male mice (ob/ob, B6.Cg-Lep<ob>/J and db/db, C57BLKS/JIar-+Leprdb/+Leprdb). All animals were allowed free access to normallaboratory chow and water. Except normal C57BL/6 mice, ob/ob and db/dbmice were used when average blood glucose levels were ≥300 mg/dL 3-5days before administration.

Blood glucose values were measured by tail bleeding from non-fastedanimals using glucometer (Optium™ Xceed™ Diabetes Monitoring System,Abbott) at 0 hr (before administration), 6^(th) hr, 18^(th) hr, 30^(th)hr, 42^(th) hr, 54^(th) hr and 66^(th) hr after ES135 treatment. Serumglucose and the percentage of post-treatment relative to pre-treatmentgroup values obtained was calculated and two-way ANOVA followed byBonferroni test was then applied for comparison between treated andvehicle groups. Differences was considered significant at *P<0.05,**P<0.01 and ***P<0.001 vs vehicle control.

While the foregoing written description of the invention enables one ofordinary skill in the art to make and use what is considered presentlyto be the best mode thereof, those of ordinary skill in the art willunderstand and appreciate the existence of variations, combinations, andequivalents of the specific embodiments, methods, and examples herein.The invention should therefore not be limited by the above describedembodiments, methods, and examples, but by all embodiments and methodswithin the scope and spirit of the invention.

What is claimed is:
 1. Use of a protein having an amino acid sequence asset forth in SEQ ID NO: 1 in manufacturing a medicament for preventingor treating a metabolic disorder in a subject.
 2. The use according toclaim 1, wherein the metabolic disorder is selected from the groupconsisting of hyperglycemia, impaired fasting glucose, impaired glucosetolerance, insulin resistance, hyperinsulinemia, type I diabetes, typeII diabetes, refractory diabetes, and combinations thereof.
 3. The useaccording to claim 1, wherein in the medicament is formulated forsubcutaneous, topical, intraneural, intraperitoneal, intravenous,intramuscular, intracerebroventricular, or intrathecal administration.4. A method for preventing or treating a metabolic disorder, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a protein having an amino acid sequence of SEQ ID NO:
 1. 5.The method according to claim 4, wherein the protein is administeredsubcutaneously, topically, intraneurally, intraperitoneally,intravenously, intramuscularly, intracerebroventricularly orintrathecally.
 6. The method according to claim 5, wherein the proteinis administered subcutaneously.
 7. The method according to claim 4,wherein the metabolic disorder is selected from the group consisting ofhyperglycemia, impaired fasting glucose, impaired glucose tolerance,insulin resistance, hyperinsulinemia, type I diabetes, type II diabetes,refractory diabetes, and combinations thereof.
 8. The method accordingto claim 4, wherein the protein is administered at a dose of 0.01 to 1mg/kg once or twice a day.
 9. A protein having an amino acid sequence asset forth in SEQ ID NO: 1, for use in the prevention or treatment of ametabolic disorder in a subject.